Figure 1.
Tex1 structure and expression dynamics on transcriptional and protein level.
A) Schematic representation of Tex1. Black dotted: intrinsically unstructured region P27A; Black: coiled coil domain P27; grey: RING motif. B) Western Blot analysis of antibodies specific for P27A and P27 on the pellet fraction after saponin lysis of mixed stage parasite (M) and late stage parasite (LS). The late stage parasites were fractionated into a supernatant (SN) and a pellet (P) fraction after saponin lysis. M: marker C) Abundance of tex1 transcripts by gRT-PCR. RNA was isolated from tightly synchronized culture in a 4h interval (table 1). Tex1 transcript levels (grey bars) were normalized to the transcript abundance of the constitutively expressed glutaminyl-tRNA synthetase (PF13_0170). As a control for the level of synchronization msp8 transcripts were measured and compared to the PF13_0170 transcript level (white bars). D) Protein level was analyzed throughout the intraerythrocytic development cycle in a 4 h interval by Western Blot analysis using antibodies against P27A and compared to protein abundance of MSP1. The parasite age (in hours post infection) and the parasite stages (confirmed by Giemsa staining) corresponding to the time points of harvest are illustrated in table 1.
Table 1.
Time points of harvest of synchronized 3D7 in vitro culture.
Figure 2.
Immunofluorescence staining of erythrocytes infected by P. falciparum (ring, trophozoites and schizont stages) using P27-specific polyclonal rabbit sera.
P27-specific polyclonal rabbit sera was used to detect Tex1 (green) A) in late ring stages B) in trophozoite stages C) in schizont stages. Nucleus stained with DAPI (blue), transmission picture of the infected red blood cell (DIC) and merged picture of the two signals or the signals merged with transmission picture (merge), Scale bar: 5 µm.
Figure 3.
Co-localization of Tex1 with Rex1.
P27-specific polyclonal mouse sera (in red) was used to detect TEX1. Rex1 polyclonal rabbit sera (in green). (A) Ring stage parasites; (B) trophozoite stages; (C) schizont stages. Scatter plots show the degree of co-localization of the Tex1 with Rex1 signal. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.
Figure 4.
Co-localization of Tex1 with SBP1.
P27-specific polyclonal rabbit sera was used to detect Tex1 (red). Co-localization was performed using SBP1 polyclonal mouse sera (green). Co-localization was performed in ring (A) trophozoite (B) and schizont stage (C) infected RBCs. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.
Figure 5.
Co-localization of Tex1 with MAHRP1.
P27-specific polyclonal rabbit sera was used to detect Tex1 (red). Co-localization was performed using MAHRP1 polyclonal mouse sera (green). Co-localization was performed in ring stage (A) trophozoite (B) and schizont stage (C) infected RBC. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.
Figure 6.
Tex1 localization in trophozoite and schizont stages with respect to newly described structures called tethers.
Co-localization of Tex1 (red) with MAHRP2 (green) A) in trophozoite stages and B) in schizont stages. Nuclear DNA was stained with DAPI (blue), Transmission image (DIC), Scale bar: 5 µm.
Figure 7.
Dual solubility pattern of Tex1 shown by Western blot analysis of membrane fractionation assay of late stage parasites.
Soluble proteins from membranes of RBCs infected with late stage parasites lysed by freezing thawing cycles (lane 1). Peripheral membrane proteins extracted by sodium carbonate buffer, (lane 2). Integral membrane proteins obtained by additional 1% Triton X-100 extraction (lane 3). Insoluble proteins (remaining membrane proteins after Triton X-100 extraction (lane 4). Blot was probed with P27A-specific polyclonal rabbit sera (panel 1), anti-MAHRP1 polyclonal rabbit sera (panel 2) and SERA5 and MSP1 mouse monoclonal antibodies (panel 3).
Figure 8.
A) 3D7 infected RBC lysed with equinatoxin II. Integrity of MCs is demonstrated by the absence of the SBP1 signal after using SBP1 N-terminus specific polyclonal mouse sera (note: N-terminus of SBP1 faces the lumen of MCs). Tex1 signal on the MC surface was obtained with P27-specific polyclonal rabbit sera (in green). B) 3D7 infected RBC lysed with equinatoxin followed by Triton lysis. MC lumen is now accessible for antibodies as shown by the SBP1 signal (in red). Nuclear DNA stained with DAPI (blue), Transmission image (DIC). Scale bar: 5 µm.
Figure 9.
Brefeldin A sensitivity of Tex1 export.
3D7 infected RBC were treated with BFA and fixed (+BFA). Tex1 was stained using P27 or P27A-specific mouse antibodies (in red, upper panel: early trophozoite, middle panel: trophozoite). Tex1 visible inside the parasite in close proximity to the nucleus. A control culture (+ETOH) was incubated with equivalent concentration of ethanol, the solvent of Brefeldin A. In the control culture Tex1 was correctly exported and associated to MC (in red). The nucleus was stained with DAPI (in blue). Transmission image (DIC). scale bar: 5 µm.